Multipotent Neural Stem Cells (NSC) can be derived from adult central nervous system (CNS) tissue, embryonic stem cells (ESC), or iPSC and provide a partially committed cell population that has not exhibited evidence of tumorigenesis after long term CNS transplantation. Transplantation of NSC from these different sources has been shown by multiple investigators in different CNS injury and disease paradigms to promote recovery or ameliorate disease. Additionally, both {REDACTED} groups have shown that human NSCs transplanted in the subacute period after spinal cord injury promote functional recovery. While the role of the host immune response has been considered in the context of immune-rejection, predominantly regarding the T-cell response, the consequence of an ongoing inflammatory response within the context of the tissue microenvironment for cell fate, migration, and integration/efficacy has been largely overlooked. Critically, the tumorigeneis, fate, migration, and integration/repair potential of a stem cell is driven by: 1) the intrinsic properties of cell programming, e.g., the type and source of cell / means used to derive the cell, and maintenance/differentiation of the cell in vitro; and 2) the extrinsic factors the cell encounters. Variations in the intrinsic properties of the cell may affect the potential of that cell for uncontrolled proliferation or the response of the cell to extrinsic factors that it later encounters, defining its fate, migration, and integration/repair potential. The {REDACTED} group has demonstrated that iPS-derived neurospheres (iPS-NS) exhibit a surprisingly large degree of variation in tumorigenesis potential after CNS transplantation, which is correlated with tissue source as well as differentiation and NS forming capacity. Moreover, the intrinsic properties of hNSC populations derived from different cell sources have not been broadly characterized; in fact, {REDACTED} has published the first data in the field demonstrating the differences in fate and integration/repair potential between primary and secondary neurospheres generated via in vitro differentiation of mouse or human ESC and iPSC. In parallel, {REDACTED} has shown profound differences in the response of NSC derived from human tissue versus hESC to extrinsic signals. Together, these data suggest that both characterization of the intrinsic properties of NSCs derived from different sources is essential for our understanding of the basic biology of these cells. Investigation of molecules and signaling pathways directing hNSC fate choices in the injured CNS microenvironment will yield new insight into the mechanisms of fate and migration decisions in these cell populations.

Statement of Benefit to California:

Multipotent Neural Stem Cells (NSC) can be derived from adult central nervous system (CNS) tissue, embryonic stem cells (ESC), or induced pluripotent cells (iPSC) and provide a partially committed cell population that has not exhibited evidence of tumorigenesis after long term CNS transplantation. Transplantation of NSC from these different sources has been shown by multiple investigators in different CNS injury and disease paradigms to promote recovery or ameliorate disease. Accordingly, stem cell based therapeutics such as these have the potential to treat a variety of traumatic, congenital, and acquired human conditions. However, while much progress has been made, translational research with human stem cell populations will remain limited by the progress of the fundamental understanding of the basic biology of these cells. The {REDACTED} group has pioneered understanding the critical role of timing in considering cell transplantation therapies. More recently, this group has focused on the neural induction of mouse- and human-derived iPSC and tested the potential of these cell populations for spinal cord injury treatment in animal models. {REDACTED} has established the NOD-scid mouse as a model for experimental neurotransplantation for xenograft studies, characterizing the relationship between transplant timing, engraftment outcome, cell fate, host remyelination, and functional recovery. Recently, this group has focused on how the innate inflammatory response influences cell fate and migration. In this collaborative proposal, researchers from California and Japan propose to combine their expertise to characterize and investigate some of the most fundamental aspects of the intrinsic properties of, and extrinsic factors influencing, human induced pluripotent (hiPSC) and human embryonic (hESC) stem cells, pooling knowledge and expertise in stem cell and animal model paradigms. The experiments proposed investigate the basic cellular and molecular mechanisms underlying the role of the host environment in stem cell fate regulation, and the relationship between reprogramming and tumorigenic/fate potential of hiPS-NSC in vitro and after transplantation, and key to this collaborative effort, the interface of these two aspects of basic stem cell biology. Critically, this international collaboration combines the expertise of two of the most advanced laboratories in translational stem cell biology to address several key unresolved questions in the control of cell fate, and will promote sharing of resources, data, and techniques between these labs to advance the field. Ultimately, the collaborative work proposed may permit the development of strategies to refine cellular reprogramming techniques, alter in vitro differentiation strategies, or manipulate the microenvironment to maximize the window for potential stem cell-based neurotherapeutics.

Progress Report:

Multipotent Neural Stem Cells (NSC) can be derived from adult and fetal central nervous system (CNS) tissue, embryonic stem cells (ESC), or iPSC and provide a partially committed cell population that has not exhibited evidence of tumorigenesis after long term CNS transplantation. Transplantation of NSC from these different sources has been shown by multiple investigators in different CNS injury and disease paradigms to promote recovery or ameliorate disease. Additionally, both Dr. Okano and Dr. Anderson’s groups have shown that human NSCs transplanted in the subacute period after spinal cord injury promote functional recovery. While the role of the host immune response has been considered in the context of immune-rejection, predominantly regarding the T-cell response, the consequence of an ongoing inflammatory response within the context of the tissue microenvironment for cell fate, migration, and integration/efficacy has been largely overlooked. Critically, the tumorigeneis, fate, migration, and integration/repair potential of a stem cell is driven by: 1) the intrinsic properties of cell programming, e.g., the type and source of cell / means used to derive the cell, and maintenance/differentiation of the cell in vitro; and 2) the extrinsic factors the cell encounters. Variations in the intrinsic properties of the cell may affect the potential of that cell for uncontrolled proliferation or the response of the cell to extrinsic factors that it later encounters, defining its fate, migration, and integration/repair potential. The Nakamura/Okano group has demonstrated that iPS-derived neurospheres (iPS-NS) exhibit a surprisingly large degree of variation in tumorigenesis potential after CNS transplantation, which is correlated with tissue source as well as differentiation and NS forming capacity. Moreover, the intrinsic properties of hNSC populations derived from different cell sources have not been broadly characterized; in fact, Dr. Okano’s group has published the first data in the field demonstrating the differences in fate and integration/repair potential between primary and secondary neurospheres generated via in vitro differentiation of mouse or human ESC and iPSC. In parallel, Dr. Anderson’s group has shown profound differences in the response of NSC derived from human fetal tissue versus hESC to extrinsic signals. Together, these data suggest that both characterization of the intrinsic properties of NSCs derived from different sources is essential for our understanding of the basic biology of these cells. Investigation of molecules and signaling pathways directing hNSC fate choices in the injured CNS microenvironment will yield new insight into the mechanisms of fate and migration decisions in these cell populations.
Progress has been excellent in the first year, as has communication between the groups.
The Nakamura/Okada/Okano laboratory has regularly shared and updated us on these important findings and the progress of Aim 1 at Keio University via emails, live phone conferences and face-to-face meetings. The latest meeting occurred at the International Stem Cell Meeting in Toronto (ISSCR, June 2011), where safety and efficacy data of the initial screenings of numerous hiPS cell lines are shared and discussed which will have a significant impact on which cell lines we will work with under Aims 2 and 3.
Additionally, the Anderson laboratory took the additional step of focusing on xeno-free cells for this grant, with the goal of advancing future knowledge of utility for clinical translation based on CIRM funding. Xeno-free cells are cells that are cultured under conditions in which they are not exposed to animal proteins. Towards this goal, we have successfully transitioned multiple ESC and iPSC lines to xeno-free conditions for both maintenance, and successfully differentiated these lines to a neural stem cell lineage under parallel conditions. Moreover, by taking this step we have significantly enhanced the comparability of different cell lines for intrinsic properties and extrinsic influences, enhancing the potential impact of this work in increasing our basic understanding of stem cell biology, and how to harness it. Finally, we have conducted the first of our experiments testing the role of cell intrinsic properties in defining responses to the in vitro and in vivo microenvironment. Our data suggest that there are clear differences in intrinsic properties between cell lines, consistent with our initial hypothesis.

Although the role of the host immune response has been considered in the context of immune-rejection, predominantly regarding the T-cell response, the consequence of an ongoing inflammatory response within the context of the tissue microenvironment for cell fate, migration, and integration/efficacy has been largely overlooked. While classical immunosuppressants alter the T-cell response, these drugs have minimal impact on other immune cells such as neutrophils (polymorphonuclear (PMN) leukocytes) and macrophages (MACs)/microglia, which makes up a significant part of the host environment after traumatic injuries to the CNS, such as spinal cord injury (SCI). Accordingly, there is little known about the basic biology of either the host microenvironment or inflammatory microenvironment in influencing and interacting with either endogenous or transplanted stem cell populations. Understanding the molecules and signaling pathways directing hNSC fate choices in the injured CNS microenvironment is critical. hNSC derived from hiPS-NSC and hESC will be tested. We have therefore established and characterized hiPS-NSC and hES-NSC derived from multiple origins and tested the specific role of innate inflammatory cells (i.e. PMNs and macrophages) and molecules in cell fate, migration and proliferation of these hiPS-NSC and hES-NSC lines in vitro. Thus far, these data have revealed clear cell line specific intrinsic differences in response to inflammatory factors, which we will further investigated in the coming funding period both in vitro and in vivo.

The fate, migration, and repair potential of a stem cell is driven by a combination of intrinsic properties, such as the type, source, and maintenance/differentiation of the cell in vitro, as well as extrinsic factors the cell encounters in the in vivo environment, such as proteins related to inflammation or the growth matrix. Variations in the intrinsic properties of the cell may affect the potential of that cell for uncontrolled proliferation or the response of the cell to extrinsic factors that it encounters in its environment. We have previously shown that neural stem cells derived from human fetal tissue are highly sensitive to extrinsic inflammatory signals in vitro and in vivo. In the current studies, we sought to determine whether neural stem cell populations derived from different sources respond to the same sorts of inflammatory signals, in other words, whether these extrinsic factors affect stem cells as a general principal. Accordingly, we sought to characterize the intrinsic properties of neural stem cells derived from different sources and exposed to extrinsic inflammatory signals, including human embryonic and induced pluripotent cell, as an essential component of understanding of basic stem cell biology. We found that, in fact, all neural stem cells derived from embryonic and induced pluripotent populations responded to inflammatory signals. However, we also found that cell line intrinsic properties exert a strong degree of control, in some cases resulting in opposing consequences for cell proliferation and fate. Critically, we found that in vitro characteristics of response to extrinsic inflammatory signals were predictive for the way different cell populations behaved in vivo after transplantation. These data may offer a new opportunity to screen stem cell populations in vitro for comparability and predicted in vivo translational properties, and reveal a new and critical set of interactions between intrinsic cell programming and response to the environment.